33Synthetic Biology: Parts, Devices and Applications, First Edition. Edited by Christina Smolke.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.
3
The ability to precisely manipulate genomic DNA in living cells in a site-specific
manner has revolutionized biomedical research. Site-specific genomic modifica-
tion has greatly advanced preclinical research by creating invaluable cellular and
animal models of disease and is currently in clinical trials for therapeutic applica-
tion. Zinc finger proteins (ZFPs) represent a class of proteins that can be engi-
neered to manipulate user-defined chromosomal DNA targets with a high degree
of specificity. Zinc finger nucleases (ZFNs) cause double-stranded breaks (DSBs)
at precise genomic locations that can induce deletions, insertions, transloca-
tions, and/or point mutations in the genomic DNA via endogenous DNA repair
mechanisms. ZFPs fused to recombinases or transposases act in an autonomous
manner without the need to induce toxic DSBs. This chapter represents an over-
view of ZFPs, the various methods available to researchers for engineering them,
options for genomic modifications, methods for validation of genomic modifica-
tions, an overview of options for delivery to cells, and some novel ways that zinc
fingers (ZFs) are being used for genomic alteration.
3.1 Introduction to Zinc Finger DNA-Binding
Domains and Cellular Repair Mechanisms
3.1.1 Zinc Finger Proteins
The Cys 2 -His 2 ZF domain makes up the most common DNA-binding domain
structure in eukaryotes [1]. Structural determination of ZF domains bound to
DNA has enabled rational design of proteins to bind targeted DNA sequences
[1]. Such engineered ZFP domains can be fused to other protein domains with
differing capabilities to create enzymes capable of targeted cleavage of DNA and
other targeted genomic effects [2]. ZFPs can be engineered with a high degree of
specificity for unique genomic elements [3]. This chapter mainly focuses on the
Site-Directed Genome Modification with Engineered
3.1.1 Zinc Finger Proteins
Lauren E. Woodard1,2, Daniel L. Galvan^3 , and Matthew H. Wilson1,2
(^1) Department of Veterans Affairs, Nashville, TN 37212, USA
(^2) Vanderbilt University Medical Center, Department of Medicine, Department of Pharmacology, Nashville,
TN 37232, USA
(^3) University of Texas at MD Anderson Cancer Center, Section of Nephrology, Houston, TX 77030, USA